Harnessing the Full Potential of Regulatory T Cells by mRNA-induced Antigen Specificity

Abstract Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance, already have shown clinical potential. In this study, we describe an efficient non-viral engineering method by TCR-encoding mRNA electroporation, while also exploring the potential of further promoting on-target effects disrupting endogenous TCR using a fully RNA-based CRISPR/Cas9 technology. Endogenous disruption prior to TCR-transfection resulted more than 90% blockade expression similar high (>85%) but homogenous transgenic TCR. Importantly, following antigen-specific TCR-induced stimulation, both engineered Tregs significantly increased multiple activation suppression markers. Moreover, outcompeted polyclonal suppressing effector cell proliferation performed better blocking moDC maturation, even presence antigen-activated cells. Secretome screening revealed distinct anti-inflammatory profile for TCR-Tregs. On other hand, TCR-engineered KO co-expressed cytokines together with some pro-inflammatory cytokines, qualitatively different from their Th counterparts. Hence, features is seen Tregs. conclusion, show that versatile approach can be used induce functional TCR, CRISPR/Cas9-mediated TCRs efficiency platform. Comparison between EAE HLA-transgenic mouse model now being investigated. National Multiple Sclerosis Society (RG-1612-26484)